Frozen block embedding details
Making the frozen block.
Do I need to see the tissue when I’m cutting the frozen block?
In a small skin ellipse as shown in the pictorial, we need to see the inked aspect so we know where to make our scalpel cuts. We must use very little embedding medium in the side that we have to see. When the embedding medium freezes it becomes opaque and if there is a thick layer over the tissue we will not be able to see it. On the other hand if orientation is not critical and we are only looking for an on edge embedding such as a bowel section then we can use more embedding medium on both sides.
Is flattening artifact important?
If flattening is not acceptable then make sure there is enough room under the elevated block for the tissue.
An example of this is small bowel. If one wants the villi to stand vertical then it must be placed on the griddle mucosa up, and covered with medium. Allow a bit of freezing to go on before placing the elevated freezing block. Use the elevating bars with the elevated freezing block if more room is needed.
How do I know when it’s frozen?
Give the tissue about a minute of freezing. Then very gently try to rotate the elevated freezing block a degree or so. If there is movement the block is not fully frozen yet. If it feels solid it is frozen. If you accidentally separate the blocks too early it can be easily repaired.
The fix: Just apply another squirt of embedding medium and put the elevated block on again.
Removing the frozen block
Use the plastic putty knife that comes with the system to lift the frozen block from whatever side it sticks to. Don’t use a scalpel…you might poke your eye out!
Cutting the frozen block
This is an easy task if done at the right temperature. If the frozen block is removed right away it will be close to ideal temperature and only a slight warming between the palms may be necessary. If left for a few minutes to freeze while doing other things it may get fully frozen and require more warming. Always test it by trimming away the excess medium. This is a necessary task anyway to easily fit the tissue pieces in the well. At the optimal temperature the tissue cuts with a very firm fudge like consistency. Like cutting an ice cream cake. The warming maneuver should be done in short doses and not left between the gloves for more than a few seconds. It warms faster than you think. If over warmed, press the tissue flat to the griddle again for a brief period. This is a good idea in any very thin samples on the surface of the frozen block such as a thin skin specimen. It is being held by very little frozen medium on the edge of the skin. If slightly melted, it can dislodge on cutting.
Ok this is what the scalpel is for.
Scalpels are best served by moving the blade forward and or backward along tissue with mild pressure in an even movement without trying to push it through tissue like your chopping it. Anyone who has seen a brain cutting knows this. If the frozen block is at the ideal temperature it will cut without too much effort. This will also vary with the water content of the tissue. If you find yourself having to lever through it like your using a chefs knife it is worth warming a bit more. The smoother you cut the tissue, the flatter the surface of each piece will be and the flatter the final preparation will be. That is, there will be fewer defects to plaster over. If it flies or snaps please warm it more. The added five seconds to warm it is much faster than trying to find a piece of tissue that grew wings.
“Cutting the thin and tiny”
I was able to cut a sesame seed like a skin ellipse. It took three tries. It was a good learning experience. The medium did not adhere well to the seed. The central section was no problem. I kept dislodging the longitudinal margins.
The trick: Make sure the tissue is well frozen and move the knife into the tissue in the direction of the medium. Don’t pull the tissue away from the medium with the scalpel stroke. The lesson: With the little skin ellipses, press the tissue face one last time to the griddle after any warming and always try to have the knife moving in the direction of pushing the tissue against the medium.
Putting the tissue pieces in the wells
Tissue is placed face down in the well.
-Use the epoxy coated forceps to handle frozen tissue and keep them cold in the cryostat. If you use uncoated warm steel forceps, the pieces of frozen block will stick to the forceps and make it difficult and frustrating to arrange tissues in the well.
-Leave 2 mm. of space between each piece for medium to penetrate. This is very important. If the pieces are to close together there will be deep crevices without medium between the pieces. It will be difficult to plaster that deep and when you cut the block the section will separate where the crevices are.